Candida albicans is the most prevalent fungal pathogen in humans, causing life threatening systemic infections in immuno-compromised patients. Largely due to the rampant AIDS pandemic of the past quarter of a century the fungus C. albicans has rapidly risen from a largely harmless commensal of the humans to the most prevalent fungal pathogen (Odds, F. C. (1985) Crit. Rev. Microbiol. 12, 45-93; Calderone, R. A., and Fonzi, W. A. (2001) Trends Microbiol. 9, 327-935; Berman, J., and Sudbery P. E. (2002) Nat. Rev. Genet. 3, 918-930; Gow, N. A., et al (2002) Curr. Opin. Microbial. 5, 366-371; Liu, H. (2002) Int. J. Med. Microbial. 292, 299-311). C. albicans commonly causes life-threatening systemic infections in immuno-compromised patients. A well established virulence trait of C. albicans is its ability to switch between several morphological forms such as budding yeast, pseudohyphae and true hyphae in response to environmental cues (Leberer, E., et al (1996) Proc. Natl. Acad. Sci. USA 93, 13217-13222; Lo, H. J., et al (1997) Cell 90; 939-949; Zheng, X., et al (2004) EMBO J. 23, 1845-1856.). Serum is an inducer of this switch.
The yeast-hypha switch is well documented to play important roles in penetrating host tissues and escaping from phagocytic destruction (Cutler, J. E. (1991) Annu. Rev. Microbiol. 45, 187-218; Lo, H. J., et al (1997) Cell 90; 939-949; Phan, Q. T., et al (2000) Infect. Immun. 68, 3485-3490; Bal, C., et al (2002). Mol. Microbiol. 45, 31-44), two processes generally important for pathogenesis and virulence of many microbial pathogens. Indeed, C. albicans mutants defective in the yeast-hypha transition exhibit significantly reduced virulence (Leberer, E., et al (1997) Curr. Biol. 7, 539-546; Lo, H. J., et al (1997) Cell 90; 939-949; Braun, B. R., and Johnson, A. D. (1997). Science 277, 105-105-109; Calderone, R. A., and Fonzi, W. A. (2001). Trends Microbiol. 9, 327-935; Stoldt, V. R., et al (1997) EMBO J. 16, 1982-1991; Zheng, X., et al (2004) EMBO J. 23, 1845-1856). Thus, blocking the morphological switch holds high promise for developing effective medical interventions for candidal infections. Although a variety of inducers have been reported to trigger the yeast-hypha switch under laboratory conditions, serum is undisputedly the most potent and physiologically relevant (Gow NA. (1997) Curr. Top. Med. Mycol. 8, 4355; Ernst, J. F. (2000) Microbiology 146, 1763-1774). In spite of the fact that the serum activity was first reported half a century ago (Reynolds, R., and Braude, A. I. (1956) Olin. Res. Proc 4, 40), the identity of the inducer(s) and its sensor in C. albicans remain ill defined.
Feng et al. (1999) first found that the majority of the serum hyphal inducer(s) can pass through a dialysis membrane with a molecular weight cut-off of 1 kDa (Feng, Q. et al (1999). J. Bacterial. 181, 6339-6346.). Hudson et al. (2004) recently reported that there are two distinct hyphal inducers in serum (Hudson, D. A., et al (2004) Microbiology 150, 3041-3049). Glucose was described to be a dialyzable inducer responsible for −80% of the inducing activity. A minor inducer was found to be non-dialyzable and trichloroacetic acid-precipitable. This report observed that adding the dialyzable fraction to glucose-containing medium did not induce the yeast-hypha switch.
The key signalling cascade responsible for serum-induced hyphal growth has been well established to be the cyclic AMP/protein kinase A (PKA) pathway (Leberer, E., et al (2001) Mol. Microbiol. 42, 673-687; Liu, H. (2001) Curr. Opin. Microbiol. 4, 728-735; Roche, C. R., et al (2001) Mol. Biol. Cell 12, 3631-3643). C. albicans genome contains a single adenylate cyclases gene CDC35. Hyphal induction activates this enzyme, resulting in a spike of cellular cAMP level and subsequent activation of PKA (Bahn, Y. S, and Sundstrom, P. (2001) J. Bacteriol. 183, 3211-3221; Roche, C. R., et al (2001) Mol. Biol. Cell 12, 3631-3643). This pathway leads to the activation of a transcription factor CaEfg1p which regulates the expression of a large number of hypha-specific genes (Ernst, J. F. (2000) Microbiology 146, 1763-1774; Lane, S., et al (2001) J. Biol. Chem. 276, 48988-48996; Liu, H. (2001) Curr. Opin. Microbiol. 4, 728-735). CDC35 deletion causes a complete loss of hyphal development as well as severely retarded yeast growth (Roche, C. R., et al (2001) Mol. Biol. Cell 12, 3631-3643). Although Cdc35 is thought to reside near the very beginning of the cAMP/PKA pathway, its possible role in signal sensing has not been addressed.
Cdc35 contains at least three functional domains: an N-terminal RAS-association (RA) domain, a middle domain made up of 15 LRRs and a carboxy terminal catalytic domain of adenylyl cyclase. The long LRR domain is a prominent feature of several families of proteins involved in the innate immunity in mammals, insects and plants that have evolved to recognize, a range of conserved pathogen associated molecular patterns including peptidoglycans (PG), lipopolyssacharides, lipoproteins, etc (Chamaillard, M., et al (2003). Cell Microbiol. 5, 581 592; Chamaillard, M., (2003) Nat. Rev. Immunol. 4, 702-707; Inohara, N., and Nunez, G. (2003). Nat. Rev. Immunol. 3, 371-382; Girardin, S. E., and Philpott; D. J. (2004) Eur. J. Immunol. 34, 1777-1782).
The present invention seeks to provide a hitherto unknown means for modulating hyphal growth. It provides methods for screening modulators that are capable of achieving this outcome as well as therapeutically active compounds that are able to interfere with C. albicans virulence.
General
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. The invention includes all such variation and modifications. The invention also includes all of the steps, features, formulations and compounds referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features.
Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means that it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in this text is not repeated in this text is merely for reasons of conciseness.
Any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention.
The present invention is not to be limited in scope by any of the specific embodiments described herein. These embodiments are intended for the purpose of exemplification only. Functionally equivalent products, formulations and methods are clearly within the scope of the invention as described herein.
The invention described herein may include one or more range of values (e.g. size, concentration etc). A range of values will be understood to include all values within the range, including the values defining the range, and values adjacent to the range which lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range.
Throughout this specification, unless the context requires otherwise, the word “comprise” or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. It is also noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as “comprises”, “comprised”, “comprising” and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of” and “consists essentially of” have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.
Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs.